Transgenic plants have been an integral component of advances made in agricultural biotechnology. They are necessary tools for the production of plants exhibiting desirable traits (e.g. herbicide and insect resistance, drought and cold tolerance), or producing products of nutritional or pharmaceutical importance. As the applications of transgenic plants become ever more sophisticated, it is becoming increasingly necessary to develop strategies to fine-tune the expression of introduced genes. The ability to tightly regulate the expression of transgenes is important to address many safety, regulatory and practical issues. To this end, it is necessary to develop tools and strategies to regulate the expression of transgenes in a predictable manner.
Several strategies have so far been employed to control plant gene/transgene expression. These include the use of regulated promoters, such as inducible or developmental promoters, whereby the expression of genes of interest is driven by promoters responsive to various regulatory factors (Gatz, 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol., 48: 89). Other strategies involve co-suppression (Eisner et al., 1998, Ther. Appl. Genet., 97: 801) or anti-sense technology (Kohno-Murase et al., 1994, Plant Mol. Biol., 26: 1115), whereby plants are transformed with genes, or fragments thereof, that are homologous to genes either in the sense or antisense orientations. Chimeric RNA-DNA oligonucleotides have also been used to block the expression of target genes in plants (Beetham et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 8774; Zhu et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 8768).
The ROS protein is encoded by the chromosomal gene, ROS, of Agrobacterium tumefaciens. In this organism, the ROS protein acts as a negative regulator for the expression of the Ti-plasmid-encoded VirC, VirD and IPT genes (Cooley et al., J.1991, Bacteriol. 173: 2608-2616; Chou et al., 1998, Proc. Natl. Acad. Sci., 95: 5293; Archdeacon J et al. 2000, FEMS Microbiol Let. 187: 175-178; D'Souza-Ault M. R., 1993, J Bacteriol 175: 3486-3490). The ROS protein is a DNA binding protein that is able to bind a ROS operator sequence (D'Souza-Ault M. R., 1993, J Bacteriol 175: 3486-3490).
Analysis of the amino acid sequence of the ROS protein reveals that it has a DNA binding motif of the C2H2 zinc finger configuration (Chou et al., 1998, Proc. Natl. Acad. Sci., 95: 5293). Typical zinc fingers are characterised by the presence of two cysteine and two histidine residues joined together by the coordination of a single zinc ion. A stretch of amino acids forms a peptide loop, known as the zinc finger motif that is required for DNA binding. Zinc finger proteins represent a significant portion of proteins in eukaryotes, but are rare in prokaryotes. The zinc finger of the bacterial ROS protein varies from its counterparts in eukaryotes in that the ROS protein has only one zinc finger motif, while eukaryotic zinc finger proteins have multiple zinc finger motifs. In addition, there are 9 amino acid residues making up the peptide loop spacing the zinc finger motif in the ROS protein as compared to the 12 amino acids that make up the loops of zinc fingers of eukaryotic proteins.
There is no suggestion for the use of ROS repressor to regulate gene expression within plants. The present invention provides a method for the regulation of gene expression in plants using a nucleic acid sequence, or derivatives of thereof, that encode ROS.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.